Novel compounds

ABSTRACT

Pyrimidone and pyridone compounds of the formula:  
                 
 
are inhibitors of the enzyme Lp-PLA 2  and are of use in therapy, in particular for treating atherosclerosis.

The present invention relates to certain novel pyrimidone and pyridonecompounds, processes for their preparation, intermediates useful intheir preparation, pharmaceutical compositions containing them and theiruse in therapy, in particular in the treatment of atherosclerosis.

WO 95/00649 (SmithKline Beecham plc) describes the phospholipase A₂enzyme Lipoprotein Associated Phospholipase A₂ (Lp-PLA₂), the sequence,isolation and purification thereof, isolated nucleic acids encoding theenzyme, and recombinant host cells transformed with DNA encoding theenzyme. Suggested therapeutic uses for inhibitors of the enzyme includedatherosclerosis, diabetes, rheumatoid arthritis, stroke, myocardialinfarction, reperfusion injury and acute and chronic inflammation. Asubsequent publication from the same group further describes this enzyme(Tew D et al, Arterioscler Thromb Vas Biol 1996:16;591-9) wherein it isreferred to as LDL-PLA₂. A later patent application (WO 95/09921, IcosCorporation) and a related publication in Nature (Tjoelker et al, vol374, 6 April 1995, 549) describe the enzyme PAF-AH which has essentiallythe same sequence as Lp-PLA₂ and suggest that it may have potential as atherapeutic protein for regulating pathological inflammatory events.

It has been shown that Lp-PLA₂ is responsible for the conversion ofphosphatidylcholine to lysophosphatidylcholine, during the conversion oflow density lipoprotein (LDL) to its oxidised form. The enzyme is knownto hydrolyse the sn-2 ester of the oxidised phosphatidylcholine to givelysophosphatidylcholine and an oxidatively modified fatty acid. Bothproducts of Lp-PLA₂ action are biologically active withlysophosphatidylcholine in particular having several pro-atherogenicactivities ascribed to it, including monocyte chemotaxis and inductionof endothelial dysfunction, both of which facilitate monocyte-derivedmacrophage accumulation within the artery wall. Inhibition of theLp-PLA₂ enzyme would therefore be expected to stop the build up of thesemacrophage enriched lesions (by inhibition of the formation oflysophosphatidylcholine and oxidised free fatty acids) and so be usefulin the treatment of atherosclerosis.

The increased lysophosphatidylcholine content of oxidatively modifiedLDL is also thought to be responsible for the endothelial dysfunctionobserved in patients with atherosclerosis. Inhibitors of Lp-PLA₂ couldtherefore prove beneficial in the treatment of this phenomenon. AnLp-PLA₂ inhibitor could also find utility in other disease states thatexhibit endothelial dysfunction including diabetes, hypertension, anginapectoris and after ischaemia and reperfusion.

In addition, Lp-PLA₂ inhibitors may also have a general application inany disorder that involves activated monocytes, macrophages orlymphocytes, as all of these cell types express Lp-PLA₂. Examples ofsuch disorders include psoriasis.

Furthermore, Lp-PLA₂ inhibitors may also have a general application inany disorder that involves lipid oxidation in conjunction with Lp-PLA₂activity to produce the two injurious products, lysophosphatidylcholineand oxidatively modified fatty acids. Such conditions include theaforementioned conditions atherosclerosis, diabetes, rheumatoidarthritis, stroke, myocardial infarction, ischaemia, reperfusion injuryand acute and chronic inflammation.

Patent applications WO 96/12963, WO 96/13484, WO96/19451, WO 97/02242,WO97/217675, WO 97/217676, WO 96/41098, and WO 97/41099 (SmithKlineBeecham plc) disclose inter alia various series of4-thionyl/sulfinyl/sulfonyl azetidinone compounds which are inhibitorsof the enzyme Lp-PLA₂. These are irreversible, acylating inhibitors (Tewet al, Biochemistry, 37, 10087, 1998).

A further class of compounds has now been identified which arenon-acylating inhibitors of the enzyme Lp-PLA₂. Thus, WO 99/24420, WO00/10980, WO 00/66566, WO 00/66567 and WO 00/68208 (SmithKline Beechamplc) disclose a class of pyrimidone compounds which are exemplified byan optionally substituted 2-benzylthio or 2-benzyloxy substituent. Wehave now found that the pyrimidone ring, optionally replaced by apyridone ring, may be fused to a heterocyclyl ring to give compoundshaving good activity as inhibitors of the enzyme Lp-PLA₂.

Accordingly, the present invention provides a compound of formula (I):

in which:

-   -   R¹ is an aryl group, optionally substituted by 1, 2, 3 or 4        substituents which may be the same or different selected from        C₍₁₋₆₎alkyl, C₍₁₋₆₎alkoxy, C₍₁₋₆₎alkylthio, hydroxy, halogen,        CN, and mono to perfluoro-C₍₁₋₄₎alkyl;    -   R² and R³ together with the ring carbon atoms to which they are        attached form a fused 5- or 6-membered heterocyclyl ring        containing 1 or 2 heteroatoms which may be the same or different        selected from N, O and S, optionally substituted by 1, 2 or 3        substituents which may be the same or different selected from        oxo, hydroxy, halogen, OR⁷, COR⁷, COOR⁷, CONR⁹R¹⁰, SR⁷, NR⁷COR⁸,        SO₂NR⁹R¹⁰, NR⁷SO₂R⁸, and C₍₁₋₆₎alkyl optionally substituted by        1, 2 or 3 substituents selected from hydroxy, halogen, OR⁷,        COR⁷, carboxy, COOR⁷, CONR⁹R¹⁰ and NR⁹R¹⁰;    -   R⁴ is hydrogen, C₍₁₋₆₎alkyl which may be unsubstituted or        substituted by 1, 2 or 3 substituents selected from hydroxy,        halogen, OR⁷, COR⁷, carboxy, COOR⁷, CONR⁹R¹⁰, NR⁹R¹⁰, NR⁷COR⁸,        mono- or di-(hydroxyC₍₁₋₆₎alky)amino and        N-hydroxyC₍₁₋₆₎alkyl-N—C₍₁₋₆₎alkylamino; or    -   R⁴ is Het-C₍₀₋₄₎alkyl in which Het is a 5- to 7-membered        heterocyclyl ring comprising N and optionally O or S, and in        which N may be substituted by COR⁷, COOR⁷, CONR⁹R¹⁰, or        C₍₁₋₆₎allyl optionally substituted by 1, 2 or 3 substituents        selected from hydroxy, halogen, OR⁷, COR⁷, carboxy, COOR⁷,        CONR⁹R¹⁰ and NR⁹R¹⁰, for instance, piperidin-4-yl,        pyrrolidin-3-yl;    -   R⁵ is an aryl or a heteroaryl ring optionally substituted by 1,        2, 3 or 4 substituents which may be the same or different        selected from C₍₁₋₆₎alkyl, C₍₁₋₆₎alkoxy, C₍₁₋₆₎alkylthio,        arylC₍₁₋₆₎alkoxy, hydroxy, halogen, CN, COR⁷, carboxy, COOR⁷,        NR⁷COR⁸, CONR⁹R¹⁰, SO₂NR⁹R¹⁰, NR⁷SO₂R⁸, NR⁹R¹⁰, mono to        perfluoro-C₍₁₋₄₎allyl and mono to perfluoro-C₍₁₋₄₎alkoxy;    -   R⁶ is an aryl or a heteroaryl ring which is further optionally        substituted by 1, 2, 3 or 4 substituents which may be the same        or different selected from C₍₁₋₁₈₎alkyl, C₍₁₋₁₈₎alkoxy,        C₍₁₋₆₎alkylthio, C₍₁₋₆₎alkylsulfonyl, arylC₍₁₋₆₎alkoxy, hydroxy,        halogen, CN, COR⁷, carboxy, COOR⁷, CONR⁹R¹⁰, NR⁷COR⁸, SO₂NR⁹R¹⁰,        NR⁷SO₂R⁸, NR⁹R¹⁰, mono to perfluoro-C₍₁₋₄₎alkyl and mono to        perfluoro-C₍₁₋₄₎alkoxy, or C₍₅₋₁₀₎alkyl;    -   R⁷ and R⁸ are independently hydrogen or C₍₁₋₁₂₎alkyl, for        instance C₍₁₋₄₎allyl (e.g. methyl or ethyl);    -   R⁹ and R¹⁰ which may be the same or different is each selected        from hydrogen, or C₍₁₋₁₂₎alkyl, or R⁹ and R¹⁰ together with the        nitrogen to which they are attached form a 5- to 7 membered ring        optionally containing one or more further heteroatoms selected        from oxygen, nitrogen and sulphur, and optionally substituted by        one or two substituents selected from hydroxy, oxo, C₍₁₋₄₎alkyl,        C₍₁₋₄₎alkylcarboxy, aryl, e.g. phenyl, or aralkyl, e.g benzyl,        for instance morpholine or piperazine;    -   X is C₍₂₋₄₎alkylene, optionally substituted by 1, 2 or 3        substituents selected from methyl and ethyl, CH═CH or (CH₂)_(n)S        where n is 1, 2 or 3; and    -   Y is CH or N.

In one aspect the aryl group of R¹ may be phenyl or naphthyl.Preferably, R¹ is phenyl optionally substituted by halogen, C₍₁₋₆₎alkyl,trifluoromethyl, C₍₁₋₆₎alkoxy, preferably, from 1 to 3 fluoro, morepreferably, 2,3-difluoro.

In another aspect R² and R³ together with the ring carbon atoms to whichthey are attached may form a fused 5- or 6-membered heterocyclyl ringcontaining a sulphur atom, a nitrogen atom or an oxygen atom,particularly a fused 5-membered heterocyclyl ring.

In another aspect R⁴ may be hydrogen, methyl, 2-(diethylamino)ethyl,2-(piperidin-1-yl)ethyl, 2-(pyrrolidin-1-yl)ethyl, 1-methyl-piperidinyl,1-ethyl-piperidin-4-yl, 1-ethyl-pyrrolidin-2-ylmethyl or1-(2-methoxyethyl)piperidin-4-yl. Preferably R⁴ is2-(diethylamino)ethyl, 1-ethyl-piperidin-4-yl or1-(2-methoxyethyl)piperidinyl.

In another aspect R⁵ may be phenyl or pyridyl. Preferably, R⁵ is phenyl.

In another aspect R⁶ may be phenyl optionally substituted by halogen, ortrifluoromethyl, preferably at the 4-position, or ethyl. Preferably, R⁶is phenyl substituted by trifluoromethyl at the 4-position.

Preferably, R⁵ and R⁶ together form a 4-(phenyl)phenyl or a2-(phenyl)pyridinyl substituent in which the remote phenyl ring may beoptionally substituted by halogen or trifluoromethyl, preferably at the4-position.

Preferably X is C₍₂₋₄₎alkylene, more preferably C₍₂₋₃₎alkylene, mostpreferably, (CH₂)₂.

Preferably Y is N.

It will be appreciated that compounds of the present invention maycomprise one or more chiral centres so that stereoisomers may be formed.The present invention encompasses all stereoisomers of the compounds offormula (I) including geometric isomers and optical isomers (eg.diastereoisomers and enantiomers) whether as individual stereoisomersisolated such as to be substantially free of the other stereoisomers(ie. pure) or as mixtures thereof including racemic modifications. Anindividual stereoisomer isolated such as to be substantially free ofother stereoisomer (ie. pure) will preferably be isolated such that lessthan 10% preferably less than 1% especially less than 0.1% of the otherstereoisomers is present.

Certain compounds of formula (I) may exist in one of several tautomericforms. It will be understood that the present invention encompasses alltautomers of the compounds of formula (I) whether as individualtautomers or as mixtures thereof.

It will be appreciated that in some instances, compounds of the presentinvention may include a basic function such as an amino group as asubstituent. Such basic functions may be used to form acid additionsalts, in particular pharmaceutically acceptable salts. Pharmaceuticallyacceptable salts include those described by Berge, Bighley, andMonkhouse, J. Pharm. Sci., 1977, 66, 1-19. Such salts may be formed frominorganic and organic acids. Representative examples thereof includemaleic, fumaric, benzoic, ascorbic, pamoic, succinic,bismethylenesalicylic, methanesulfonic, ethanedisulfonic, acetic,propionic, tartaric, salicylic, citric, gluconic, aspartic, stearic,palmitic, itaconic, glycolic, p-aminobenzoic, glutamic, taurocholicacid, benzenesulfonic, p-toluenesulfonic, hydrochloric, hydrobromic,sulfuric, cyclohexylsulfamic, phosphoric and nitric acids.

It will be appreciated that in some instances, compounds of the presentinvention may include a carboxy group as a substituent. Such carboxygroups may be used to form salts, in particular pharmaceuticallyacceptable salts. Pharmaceutically acceptable salts include thosedescribed by Berge, Bighley, and Monkhouse, J. Pharm. Sci., 1977, 66,1-19. Preferred salts include alkali metal salts such as the sodium andpotassium salts.

When used herein, the term “alkyl” and similar terms such as “alkoxy”includes all straight chain and branched isomers. Representativeexamples thereof include methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, iso-butyl, t-butyl, n-pentyl and n-hexyl.

When used herein, the term “aryl” refers to, unless otherwise defined, amono- or bicyclic aromatic ring system containing up to 10 carbon atomsin the ring system, for instance phenyl or naphthyl.

When used herein, the term “heteroaryl” refers to a mono- or bicyclicheteroaromatic ring system comprising up to four, preferably 1 or 2,heteroatoms each selected from oxygen, nitrogen and sulphur. Each ringmay have from 4 to 7, preferably 5 or 6, ring atoms. A bicyclicheteroaromatic ring system may include a carbocyclic ring.

When used herein, the terms “halogen” and “halo” include fluorine,chlorine, bromine and iodine and fluoro, chloro, bromo and iodo,respectively.

When used herein, the term “heterocyclyl” refers to a non-aromatic ringcomprising one or two heteroatoms.

It is to be understood that the present invention covers allcombinations of substituent groups referred to hereinabove.

A representative compound of formula (I) isN-(2-diethylaminoethyl)-2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-5,7-dihydro-4H-thieno[3,4-d]pyrimidin-1-yl)-N-4-(4-trifluoromethylphenyl)benzyl)acetamideor a pharmaceutically acceptable salt thereof, in particular thebitartrate salt.

Since the compounds of the present invention, in particular compounds offormula (I), are intended for use in pharmaceutical compositions, itwill be understood that they are each provided in substantially pureform, for example at least 50% pure, more suitably at least 75% pure andpreferably at least 95% pure (% are on a wt/wt basis). Impurepreparations of the compounds of formula (I) may be used for preparingthe more pure forms used in the pharmaceutical compositions. Althoughthe purity of intermediate compounds of the present invention is lesscritical, it will be readily understood that the substantially pure formis preferred as for the compounds of formula (I). Preferably, wheneverpossible, the compounds of the present invention are obtained incrystalline form.

When some of the compounds of this invention are allowed to crystalliseor are re-crystallised from organic solvents, solvent of crystallisationmay be present in the crystalline product. This invention includeswithin its scope such solvates. Similarly, some of the compounds of thisinvention may be crystallised or re-crystallised from solventscontaining water. In such cases water of hydration may be formed. Thisinvention includes within its scope stoichiometric hydrates as well ascompounds containing variable amounts of water that may be produced byprocesses such as lyophilisation. In addition, different crystallisationconditions may lead to the formation of different polymorphic forms ofcrystalline products. This invention includes within its scope allpolymorphic forms of the compounds of formula (I).

Compounds of the present invention are inhibitors of the enzymelipoprotein associated phospholipase A₂ (Lp-PLA₂) and as such areexpected to be of use in therapy, in particular in the treatment ofatherosclerosis. In a further aspect therefore the present inventionprovides a compound of formula (I) for use in therapy.

The compounds of formula (I) are inhibitors of lysophosphatidylcholineproduction by Lp-PLA₂ and may therefore also have a general applicationin any disorder that involves endothelial dysfunction, for exampleatherosclerosis, diabetes, hypertension, angina pectoris and afterischaemia and reperfusion. In addition, compounds of formula (I) mayhave a general application in any disorder that involves lipid oxidationin conjunction with enzyme activity, for example in addition toconditions such as atherosclerosis and diabetes, other conditions suchas rheumatoid arthritis, stroke, inflammatory conditions of the brainsuch as Alzheimer's Disease, myocardial infarction, reperfusion injury,sepsis, and acute and chronic inflammation.

Further applications include any disorder that involves activatedmonocytes, macrophages or lymphocytes, as all of these cell typesexpress Lp-PLA₂. Examples of such disorders include psoriasis.

Accordingly, in a further aspect, the present invention provides for amethod of treating a disease state associated with activity of theenzyme Lp-PLA₂ which method involves treating a patient in need thereofwith a therapeutically effective amount of an inhibitor of the enzyme.The disease state may be associated with the increased involvement ofmonocytes, macrophages or lymphocytes; with the formation oflysophosphatidylcholine and oxidised free fatty acids; with lipidoxidation in conjunction with Lp PLA₂ activity; with ischemia andreperfusion; or with endothelial dysfunction.

Compounds of the present invention may also be of use in treating theabove mentioned disease states in combination with ananti-hyperlipidaemic, anti-atherosclerotic, anti-diabetic, anti-anginal,anti-inflammatory, or anti-hypertension agent or an agent for loweringLp(a). Examples of the above include cholesterol synthesis inhibitorssuch as statins, anti-oxidants such as probucol, insulin sensitisers,calcium channel antagonists, and anti-inflammatory drugs such as NSAIDs.Examples of agents for lowering Lp(a) include the aminophosphonatesdescribed in WO 97/02037, WO 98/28310, WO 98/28311 and WO 98/28312(Symphar SA and SmithKline Beecham).

A preferred combination therapy will be the use of a compound of thepresent invention and a statin. The statins are a well known class ofcholesterol lowering agents and include atorvastatin, simvarstatin,pravastatin, cerivastatin, fluvastatin, lovastatin and ZD 4522 (alsoreferred to as S-4522, rosuvastatin, Astra Zeneca). The two agents maybe administered at substantially the same time or at different times,according to the discretion of the physician.

A further preferred combination therapy will be the use of a compound ofthe present invention and an anti-diabetic agent or an insulinsensitiser, as coronary heart disease is a major cause of death fordiabetics. Within this class, preferred compounds for use with acompound of the present invention include the PPARgamma activators, forinstance GI262570 (GlaxoSmithKline) and the glitazone class of compoundssuch as rosiglitazone (Avandia, GlaxoSmithKline), troglitazone andpioglitazone.

In therapeutic use, the compounds of the present invention are usuallyadministered in a standard pharmaceutical composition. The presentinvention therefore provides, in a further aspect, a pharmaceuticalcomposition comprising a compound of formula (I) and a pharmaceuticallyacceptable carrier, optionally with one or more other therapeuticcompounds such as a statin or an anti-diabetic.

Suitable pharmaceutical compositions include those which are adapted fororal or parenteral administration or as a suppository.

Compounds of formula (I) which are active when given orally can beformulated as liquids, for example syrups, suspensions or emulsions,tablets, capsules and lozenges. A liquid formulation will generallyconsist of a suspension or solution of the compound or pharmaceuticallyacceptable salt in a suitable liquid carrier(s) for example, ethanol,glycerine, non-aqueous solvent, for example polyethylene glycol, oils,or water with a suspending agent, preservative, flavouring or colouringagent. A composition in the form of a tablet can be prepared using anysuitable pharmaceutical carrier(s) routinely used for preparing solidformulations. Examples of such carriers include magnesium stearate,starch, lactose, sucrose and cellulose. A composition in the form of acapsule can be prepared using routine encapsulation procedures. Forexample, pellets containing the active ingredient can be prepared usingstandard carriers and then filled into a hard gelatin capsule;alternatively, a dispersion or suspension can be prepared using anysuitable pharmaceutical carrier(s), for example aqueous gums,celluloses, silicates or oils and the dispersion or suspension thenfilled into a soft gelatin capsule. Typical parenteral compositionsconsist of a solution or suspension of the compound of formula (I) in asterile aqueous carrier or parenterally acceptable oil, for examplepolyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil orsesame oil. Alternatively, the solution can be lyophilised and thenreconstituted with a suitable solvent just prior to administration. Atypical suppository formulation comprises a compound of formula (I)which is active when administered in this way, with a binding and/orlubricating agent such as polymeric glycols, gelatins or cocoa butter orother low melting vegetable or synthetic waxes or fats.

Preferably the composition is in unit dose form such as a tablet orcapsule. Each dosage unit for oral administration contains preferablyfrom 1 to 500 mg (and for parenteral administration contains preferablyfrom 0.1 to 25 mg) of a compound of the formula (I). The daily dosageregimen for an adult patient may be, for example, an oral dose ofbetween 1 mg and 1000 mg, preferably between 1 mg and 500 mg, or anintravenous, subcutaneous, or intramuscular dose of between 0.1 mg and100 mg, preferably between 0.1 mg and 25 mg, of the compound of theformula (I), the compound being administered 1 to 4 times per day.Suitably the compounds will be administered for a period of continuoustherapy, for example for a week or more.

According to a first process (A), a compound of formula (I) may beprepared by reacting an acid compound of formula (II):

in which X, Y, R¹, R² and R³ are as hereinbefore defined,with an amine compound of formula (III):R⁶—R⁵—CH₂NHR⁴  (III)in which R⁴, R⁵ and R⁶ are as hereinbefore defined; under amide formingconditions.

Suitable amide forming conditions are well known in the art and includetreating the acid of formula (II) with the amine of formula (III) in thepresence of a coupling agent such as1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide (DEC).

A compound of formula (II) may be readily prepared from a correspondingunsubstituted compound of formula (IV):

in which X, Y, R¹, R² and R³ are as hereinbefore defined,by reaction with a compound of formula (V):LCH₂CO₂R¹¹  (V)in which L is a leaving group such as trifluoromethanesulphonate orhalo, for example, chloro, bromo or iodo, and R¹¹ is C₍₁₋₆₎alkyl, forexample ethyl or t-butyl, in the presence of a base such as a tertiaryamine, for example di-isopropylethylamine; to form an intermediate ester(VI),

in which X, Y, R¹, R², R³ and R¹¹ are as hereinbefore defined,and thereafter, removing R¹¹ by treating with a de-esterifying agent,for instance, for t-butyl, trifluoroacetic acid.

It will be appreciated that removal of R¹¹ may be carried out as aseparate step, so that an acid of formula (II), or a salt thereof, forexample the sodium salt, is isolated or, alternatively, that the acid offormula (II), or a salt thereof, is formed from the intermediate ester(VI), prior to reaction with an amine of formula (III).

Thus according to a further process B, a compound of formula (I) may beprepared by (a) treating a compound of formula (VI) with ade-esterifying agent to form a compound of formula (II) and (b) reactingsaid compound of formula (II) with an amine of formula (III), underamide forming conditions.

In a further aspect, process B may include as a preliminary step (a)reacting a compound of formula (IV) with a compound of formula (V), toform the intermediate ester (VI), which need not be isolated prior totreatment with the de-esterifying agent in step (a).

When Y is N, the pyrimidone of formula (IV) may be readily prepared byadapting a standard pyrimidone synthesis involving an amidine and a1,3-dicarbonyl compound, by reacting an amidine of formula (VII):

in which R¹ and X are as hereinbefore defined,preferably as a salt thereof, for instance the hydrochloride salt,with a compound of formula (VIII):

in which R² and R³ are as hereinbefore defined.

Alternatively, for pyrimidones in which X is (CH₂)_(n)S, theintermediate compound of formula (IV) may be formed by reacting acompound of formula (VIII) with thiourea in the presence of sodiumethoxide (preferably generated in situ from sodium and ethanol),followed by alkylation with R¹L in which R¹ and L are as hereinbeforedescribed. Conditions for the alkylation reaction typically includethioether forming conditions. Advantageously, the reaction is carriedout in the presence of a base such as sodium ethoxide or potassiumcarbonate, preferably in a solvent such as ethanol or dimethylformamide,or a secondary or tertiary amine base such as di-isopropylethylamine, ina solvent such as dichloromethane.

When Y is CH, the overall synthesis of the compounds of formula (I) isillustrated in the following scheme:

Referring to the scheme, the ester (VI) is usually prepared by N-1alkylation of (IV) using (V) in which R¹¹ as hereinbefore defined, e.g.(V) is t-butyl bromoacetate or ethyl bromoacetate, in the presence of abase e.g. BuLi in THF or diisopropylethylamine in dichloromethane (stepc).

When X is CH₂S, the R¹X substituent is preferably introduced bydisplacement of a leaving group L² (e.g. Cl) (step e) on a pyridine (X),to give the 2-substituted pyridine (IX). Transformation of (IX) to the4-pyridone (IV) is accomplished by deprotection of the 4-oxygen (e.g.using Ph₃P)₃RhCl when in aq. ethanol when R¹²=allyl) (step d). Thepyridine (X) may be prepared by steps (i), (h), (g) and (f), in which:

-   (f) treatment of (XP with R¹²OH (XI), in which R¹² is e.g.    C₍₁₋₆₎alkyl or allyl, and sodium hydride in DMF;-   (g) treatment of (XIII) with phosphorus oxychloride;-   (h) treatment of (XIV) with aq HCl with heating;-   (i) treatment of (XV) with di-lower alkyl malonate and sodium    alkoxide in alcohol (in which R¹³ is C₍₁₋₆₎allyl, typically R¹³=Et);    and-   R¹—CH₂SH (XIX) is typically prepared from the thioacetate, which is    formed from the corresponding alkyl bromide R₁—CH₂Br.

When X is alkylene, it is preferable to use steps (j) and (k)(intermediates (XVI), (XVII), (XVIII)) in which the 3-ester group isremoved from intermediate (XVI) R¹⁴=C₍₁₋₆₎alkyl by heating in diphenylether where R¹⁴=tBu (step j). Intermediate (XVI) is formed from the2,6-dioxo-1,3-oxazine (XVII) and ester (XVIII) by treatment with a base(NaH) in DMF.

It will be appreciated that compounds of formula (I) may also beprepared from other compounds of formula (I) using conventionalinterconversion procedures. Thus, a process for preparing a compound offormula (I) by interconversion of another compound of formula (I)(process C) constitutes a further aspect of the present invention.

It will be appreciated by those skilled in the art that it may bedesirable to use protected derivatives of intermediates used in thepreparation of compounds of formula (I). Thus, the above processes mayrequire deprotection as an intermediate or final step to yield thedesired compound. Thus, according to another process (D), a compound offormula (I) may be prepared by subjecting a protected derivative of acompound of formula (I) to reaction to remove the protecting group orgroups present, constituting a further aspect of the present invention.

Protection and deprotection of functional groups may be effected usingconventional means. Thus, hydroxyl groups may be protected using anyconventional hydroxyl protecting group, for example, as described inProtective Groups in Organic Chemistry, Ed. J. F. W. McOmie (PlenumPress, 1973) or Protective Groups in Organic Synthesis by Theodora W.Green (John Wiley and Sons, 1991).

Examples of suitable hydroxyl protecting groups includes groups selectedfrom alkyl (e.g. t-butyl or methoxymethyl), aralkyl (e.g. benzyl,diphenylmethyl or triphenylmethyl), heterocyclic groups such astetrahydropyranyl, acyl (e.g. acetyl or benzoyl) and silyl groups suchas trialkylsilyl (e.g. t-butyldimethylsilyl). The hydroxyl protectinggroups may be removed by conventional techniques. Thus, for examplealkyl, silyl, acyl and heterocyclic groups may be removed by solvolysis,e.g. by hydrolysis under acidic or basic conditions. Aralkyl groups suchas triphenylmethyl may be similarly be removed by solvolysis, e.g. byhydrolysis under acidic conditions. Aralkyl groups such as benzyl may becleaved by hydrogenolysis in the presence of a Noble metal catalyst suchas palladium-on-charcoal.

The present invention will now be illustrated by the following example.

EXAMPLE 1N-(2-Diethylaminoethyl)-2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-5,7-dihydro-4H-thieno[3,4-d]pyrimidin-1-yl)-N-4-(4-trifluoromethyl-phenyl)benzyl)acetamidebitartrate

2-(2-(2,3-Difluorophenyl)ethyl)-5,7-dihydro-1H-thieno[3,4-d]pyrimidin-4-one

To a solution of 3-(2,3-difluorophenyl)-propionamidine hydrochloride(0.627 g, 2.84 mmol) (prepared according to the general procedure ofAndrews et al., Mol. Cryst. Liq. Cryst., 1985, 123, 257-270) in ethanol(10 ml) was added sodium hydride (0.12 g, 3 mmol, 60% in paraffin)portionwise. After stirring for 15 min,methyl-4-oxo-tetrahydrothienyl-3-carboxylate (0.45 g, 2.8 mmol)(Maybridge Chemical Co. Ltd.) was added and the solution heated atreflux for 18 h. The solution was allowed to cool then concentrated andthe residues chromatographed over silica eluting with dichloromethanefollowed by ethyl acetate to yield the title compound as a cream solid(0.379 g, 45%). ¹H-NMR (d₆-DMSO): δ 2.85 (2H, t), 3.05 (2H, t), 3.92(2H, br), 4.08 (2H, br), 7.11 (2H, m), 7.3 (1H, m), 12.6 (1H, br s). MS(APCI+) found (M+1)=295; C₁₄H₁₂F₂N₂OS requires 294Ethyl(2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-5,7-dihydro-4H-thieno[3,4-d]pyrimidin-1-yl)acetate

A solution of2-(2-(2,3-difluorophenyl)ethyl)-5,7-dihydro-1H-thieno[3,4-d]pyrimidin-4-one(0.7 g, 2.38 mmol), ethyl bromoacetate (1.19 g, 7.14 mmol) anddiisopropylethylamine (1.24 ml, 7.14 mmol) in dichloromethane (15 ml)was stirred for 5 days, washed with 2M hydrochloric acid solution thendried MgSO₄) and concentrated. Chromatography of the residues oversilica eluting with a gradient from dichloromethane todichloromethane/ether 4:1 yielded the title compound (0.10 g, 11%).¹H-NMR (d₆-DMSO): δ 1.23 (3H, t), 2.35 (1H, t), 2.78 (1H, t), 2.9-3.1(2H, m), 3.92 (1H, br), 4.14.3 (6H, br m), 5.07 (2H, s), 7.2 (3H, m). MS(APCI+) found (M+1)=381; C₁₈H₁₈F₂N₂O₃S requires 380.(2-(2-(2,3-Difluorophenyl)ethyl)-4-oxo-5,7-dihydro-4H-thieno[3,4-d]pyrimidin-1-yl)aceticacid

A solution ofethyl(2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-5,7-dihydro-4H-thieno[3,4-d]pyrimidin-1-yl)acetate(0.1 g, 0.26 mmol) and sodium hydroxide (0.015 g, 0.37 mmol) in 1:1dioxan/water (4 ml) was stirred for 3 h then acidified with 2Mhydrochloric acid and concentrated. The residues were washed with waterthen dichloromethane and then taken up in acetone and dried (MgSO₄) andconcentrated to yield the title compound (33 mg, 36%). ¹H-NMR (d₆-DMSO):δ 3.0(4H, m), 3.8 (2×, br), 4.2 (2H, br), 4.8 (2×, s), 7.0-7.3 (4H, m).MS (APCI−) found (M−1)=351; C₁₆H₁₄F₂N₂O₃S requires 352.

N-(2-Diethylaminoethyl)-2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-5,7-dihydro-4H-thieno[3,4-d]pyrimidin-1-yl)-N-4-(4-trifluoromethylphenyl)benzyl)acetamidebitartrate

A solution of(2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-5,7-dihydro-4H-thieno[3,4-d]pyrimidin-1-yl)aceticacid (0.03 g, 0.09 mmol),N-(2-diethylaminoethyl)-4-(4-trifluoromethylphenyl)benzylamine (WO00/66567) (0.03 g, 0.09 mmol) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (DEC) (0.032 g, 0.17 mmol)in dimethylformamide (2 ml) was stirred for 18 h then concentrated. Theresidues were separated between ethyl acetate and sodium bicarbonatesolution and the organics isolated, dried (MgSO₄) and concentrated toyield the amide. The bitartrate salt was formed by dissolving the amidewith tartaric acid (0.012 g, 0.09 mmol) in methanol and concentratingthe solution to provide the title compound as the bitartrate salt (0.067g, 100%).

Free Base Data:

¹H-NMR (d₆-DMSO): δ 0.96 (6H, t), 2.2-2.7 (10H, m), 2.8-4.3 (811, m),5.2 (2H, m), 7.1 (2H, m), 7.25 (1H, m), 7.5 (2H, d), 7.65 (2H, d), 7.8(2H, d), 7.9 (2H, d). MS (APCI−) found (M−1)=683; C₃₆H₃₇F₅N₄O₂S requires684.

Bitartrate Data:

¹H-NMR (d₄-MeOH): (selected peaks) δ 1.0-1.4 (6H, m), 4.37 (2H, m),6.9-7.2 (3H, m), 7.35-7.9 (8H, m).

Biological Data

1. Screen for Lp-PLA₂ Inhibition.

Enzyme activity was determined by measuring the rate of turnover of theartificial substrate (A) at 37° C. in 50 mM HEPES(N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid) buffer containing150 mM NaCl, pH 7.4.

Assays were performed in 96 well titre plates.

Recombinant Lp-PLA₂ was purified to homogeneity from baculovirusinfected Sf9 cells, using a zinc chelating column, blue sepharoseaffinity chromatography and an anion exchange column. Followingpurification and ultrafiltration, the enzyme was stored at 6 mg/ml at 4°C. Assay plates of compound or vehicle plus buffer were set up usingautomated robotics to a volume of 170 μl. The reaction was initiated bythe addition of 20 μl of 10× substrate (A) to give a final substrateconcentration of 20 μM and 10 pd of diluted enzyme to an approximatefinal 0.1 nM Lp-PLA₂. The reaction was followed at 405 nm and 37° C. for20 minutes using a plate reader with automatic mixing. The rate ofreaction was measured as the rate of change of absorbance.

Results

The compounds described in the Examples were tested as described aboveand had IC₅₀ values in the range <0.1 to 100 nM.

1. A compound of formula (I):

in which: R¹ is an aryl group, optionally substituted by 1, 2, 3 or 4substituents which may be the same or different selected from the groupconsisting of C₍₁₋₆₎alkyl, C₍₁₋₆₎alkoxy, C₍₁₋₆₎alkylthio, hydroxy,halogen, CN, and mono to perfluoro-C₍₁₋₄₎alkyl; R² and R³ together withthe ring carbon atoms to which they are attached form a fused 5- or6-membered non-aromatic heterocyclyl ring containing 1 or 2 heteroatomswhich may be the same or different selected from N, O and S, optionallysubstituted by 1, 2 or 3 substituents which may be the same or differentselected from the group consisting of oxo, hydroxy, halogen, OR⁷, COR⁷,COOR⁷, CONR⁹R¹⁰, SR⁷, NR⁷COR⁸, SO₂NR⁹R¹⁰, NR⁷SO₂R⁸, and C₍₁₋₆₎alkyloptionally substituted by 1, 2 or 3 substituents selected from the groupconsisting of hydroxy, halogen, OR⁷, COR⁷, carboxy, COOR⁷, CONR⁹R¹⁰ andNR⁹R¹⁰; R⁴ is hydrogen, C₍₁₋₆₎alkyl which may be unsubstituted orsubstituted by 1, 2 or 3 substituents selected from the group consistingof hydroxy, halogen, OR⁷, COR⁷, carboxy, COOR⁷, CONR⁹R¹⁰, NR⁹R¹⁰,NR⁷COR⁸, mono- or di-(hydroxyC₍₁₋₆₎alkyl)amino andN-hydroxyC₍₁₋₆₎alkyl-N—C₍₁₋₆₎alkylamino; or R⁴ is Het-C₍₀₋₄₎alkyl inwhich Het is a 5- to 7-membered heterocyclyl ring comprising N andoptionally O or S, and in which N may be substituted by COR⁷, COOR⁷,CONR⁹R¹⁰, or C₍₁₋₆₎alkyl optionally substituted by 1, 2 or 3substituents selected from the group consisting of hydroxy, halogen,OR⁷, COR⁷, carboxy, COOR⁷, CONR⁹R¹⁰ and NR⁹R¹⁰; R⁵ is an aryl or aheteroaryl ring optionally substituted by 1, 2, 3 or 4 substituentswhich may be the same or different selected from the group consisting ofC₍₁₋₆₎alkyl, C₍₁₋₆₎alkoxy, C₍₁₋₆₎alkylthio, arylC₍₁₋₆₎alkoxy, hydroxy,halogen, CN, COR⁷, carboxy, COOR⁷, NR⁷COR⁸, CONR⁹R¹⁰, SO₂NR⁹R¹⁰,NR⁷SO₂R⁸, NR⁹R¹⁰, mono to perfluoro-C₍₁₋₄₎alkyl and mono toperfluoro-C₍₁₋₄₎alkoxy; R⁶ is an aryl or a heteroaryl ring which isfurther optionally substituted by 1, 2, 3 or 4 substituents which may bethe same or different selected from the group consisting ofC₍₁₋₁₈₎alkyl, C₍₁₋₁₈₎alkoxy, C₍₁₋₆₎alkylthio, C₍₁₋₆₎alkylsulfonyl,arylC₍₁₋₆₎alkoxy, hydroxy, halogen, CN, COR⁷, carboxy, COOR⁷, CONR⁹R¹⁰,NR⁷COR⁸, SO₂NR⁹R¹⁰, NR⁷SO₂R⁸, NR⁹R¹⁰, mono to perfluoro-C₍₁₋₄₎alkyl andmono to perfluoro-C₍₁₋₄₎alkoxy, or C₍₅₋₁₀₎alkyl; R⁷ and R⁸ areindependently hydrogen or C₍₁₋₁₂₎alkyl; R⁹ and R¹⁰ which may be the sameor different is each selected from the group consisting of hydrogen, orC₍₁₋₁₂₎alkyl, or R⁹ and R¹⁰ together with the nitrogen to which they areattached form a 5- to 7 membered ring optionally containing one or morefurther heteroatoms selected from the group consisting of oxygen,nitrogen and sulphur, and optionally substituted by one or twosubstituents selected from hydroxy, oxo, C₍₁₋₄₎alkyl,C₍₁₋₄₎alkylcarboxy, aryl, e.g. phenyl, or aralkyl; X is C₍₂₋₄₎alkylene,optionally substituted by 1, 2 or 3 substituents selected from the groupconsisting of methyl and ethyl, CH═CH or (CH₂)_(n)S where n is 1, 2 or3; and Y is CH or N; and pharmaceutically acceptable salts thereof.
 2. Acompound according to claim 1 wherein R¹ is phenyl optionallysubstituted by halogen, C₍₁₋₆₎alkyl, trifluoromethyl or C₍₁₋₆₎alkoxy. 3.A compound according to claim 1 wherein R² and R³ together with the ringcarbon atoms to which they are attached form a fused non-aromatic 5- or6-membered heterocyclyl ring containing a sulphur atom, a nitrogen atomor an oxygen atom.
 4. A compound according to claim 1 wherein R⁴ ishydrogen, methyl, 2-(diethylamino)ethyl, 2-(piperidin-1-yl)ethyl,2-(pyrrolidin-1-yl)ethyl, 1-methyl-piperidinyl, 1-ethyl-piperidin-4-yl,1-ethyl-pyrrolidin-2-ylmethyl or 1-(2-methoxyethyl)piperidin-4-yl.
 5. Acompound according to claim 1 wherein R⁵ is phenyl.
 6. A compoundaccording to claim 1 wherein R⁶ is phenyl optionally substituted byhalogen or trifluoromethyl.
 7. A compound according to claim 1 wherein Xis C₍₂₋₄₎alkylene.
 8. A compound according to claim 1 wherein Y is N. 9.A compound according to claim 1 which isN-(2-Diethylaminoethyl)-2-(2-(2,3-difluorophenyl)ethyl)-4-oxo-5,7-dihydro-4H-thieno[3,4-d]pyrimidin-1-yl)-N-4-(4-trifluoromethylphenyl)benzyl)acetamide.10. A pharmaceutical composition comprising a compound of formula (I) asclaimed in claim 1 and a pharmaceutically acceptable carrier. 11.(Deleted)
 12. (Deleted)
 13. A method of treating a disease stateassociated with activity of the enzyme Lp-PLA₂ which method involvestreating a patient in need thereof with a therapeutically effectiveamount of a compound of formula (I) as claimed in claim
 1. 14. A processfor preparing a compound of formula (I) as defined in claim 1 whichprocess comprises reacting an acid compound of formula (II):

in which X, Y, R¹, R² and R³ are as hereinbefore defined, with an aminecompound of formula (III):R⁶—R⁵—CH₂NHR⁴  (III) in which R⁴, R⁵ and R⁶ are as hereinbefore defined;under amide forming conditions.